The use of highly active antiretroviral therapy (HAART) against HIV-1 for the last two decades reduced mortality of the patients via extension of non-symptomatic phase of infection. The current drug compositions are not able to completely eliminate virus, since HIV-1 is capable of maintaining latent infection in stable reservoirs such as resting CD4+ T cells, naive T cells and CD34+ multipotent hematopoietic stem cells. Thus, interruption of the therapy leads to the rapid increase of viral population, whereas a long combinational ART treatment has negative side effects including neurocognitive abnormalities and cardiovascular diseases. Eradication of latent HIV-1 reservoirs could eliminate infection and, therefore, stop ART treatment of the patients. Quantitative assessment of the pool of resting CD4+ memory T cells in HAART-treated patients is essential for determination of strategies to eradicate the latent HIV-1 infection. However, a high variability in the sensitivity of existing assays such as viral outgrowth assays (VOA) and quantitative PCR-based analyses points for a pressing need to develop unified method to rapidly and precisely quantify latent HIV-1 reservoir. In this application, we propose to develop a highly-efficient method to quantitatively assess latent HIV-1 reservoirs in non-viremic HIV-1 patients via the combination of (i) RCR-based detection of circulating viral TAR RNA to diagnose the reservoir and (ii) improve VOA followed by the analysis of infectivity of Nanotrap particle-captured HIV-1 to quantify the functionally competent proviral copies. To pursue these aims we propose to use innovative Nanotrap technology originally developed to concentrate and preserve low abundance proteins, peptides, nucleic acids and whole viruses from complex biofluid matrices for diagnostic purposes. The Nanotrap hydrogel particles of about 800 nm in size will be used to capture and concentrate both the exosome-packaged circulating TAR RNA in patients' plasma samples (aim 1) and progeny virus produced by reactivated CD4+ T cells from TAR-positive patients in VOA to subsequently use nanoparticle-bound virus for infection of the reporter cells in order to quantify initial replication competent proviral copies (aim 2). These aims are supported by recent data from our group which indicated that 1) serum samples from aviremic HIV-1 patients contain detectable amounts of non-coding HIV-1 TAR RNA, suggesting the presence of transcriptionally-competent provirus in cells; 2) the extracellular TAR RNA is present in exosomes and can be effectively concentrated from serum using nanoparticles; and 3) the effective concentration of HIV-1 virions from supernatants by nanoparticles allows for enrichment of fully infectious HIV-1 that can be further quantified using functional reporter assay. We expect that results of the proposed study will allow rapid assessment of latent HIV-1 reservoirs to determine strategies for reactivation of the latent HIV-1 reservoir and elimination o reinfection.